Effects of buffering intracellular free calcium on neutrophil migration through three-dimensional matrices

Repeated transient increases in intracellular free calcium levels ([Ca²⁺]_i) are required for polymorphonuclear neutrophil migration on two-dimensional surfaces coated with fibronectin or vitronectin. Cells in which [Ca²⁺]_iis buffered with quin2 become stuck on these substrates. Neutrophils migrating through the extracellular matrix in vivo encounter these and other substrates in a three-dimensional architecture that may alter the spatial distribution of adhesion receptors in contact with the matrix. In this study, we used fluorescence confocal microscopy to obtain moving three-dimensional images of neutrophils migrating through a biological tissue (human amnion) in the presence and absence of [Ca²⁺]_i-buffering with quin2. In the absence of buffering, [Ca²⁺]_itransients similar to those seen in cells migrating in two-dimensions were observed. [Ca²⁺]_i-buffered neutrophils were able to migrate into the matrix, but they became attached firmly to the substrate at the rear of the cell, resulting in a drastically elongated morphology. Immunofluorescence revealed that neutrophils adhered to regions of the matrix that contained fibronectin. RGD-containing peptides and antibodies that block integrin adhesion receptors for fibronectin and vitronectin were able to rescue the migration of quin2-treated cells through three-dimensional gels containing fibronectin and vitronectin. These data show that neutrophils migrating throughphysiologically relevant, three-dimensional matrices undergo repetitive increases in [Ca²⁺]_ithat are required for integrin-mediated detachment from the matrix. J. Cell. Physiol. 171:168–178, 1997. © 1997 Wiley-Liss, Inc.